The present invention relates to a promoter of human p27Kip1 gene and to a method of screening a compound capable of regulating activity of the promoter.
In the eukaryotic cell cycle, several positive and negative factors control the cell cycle progression. Among the positive factors, the protein kinase family plays an important role. Each member of the family comprises a regulatory subunit, or cyclin, and a catalytic subunit named cyclin-dependent kinase (cdk). A number of reports have suggested that cyclin D-cdk4, cyclin D-cdk6, and cyclin E-cdk2 play important roles in promoting the transition from the G1 phase to the S phase by the phosphorylation of retinoblastoma protein (pRB). Recently, one further level of control has become apparent, namely the expression of cdk inhibitors (Sherr, C. J. and Roberts, J. M. (1995) Genes and Dev. 9:1149-1163). Two families of cdk inhibitor with different modes of action have already been identified in mammalian cells. One group, comprised of related proteins known as p21Cip1, p27kip1, and p57Kip2, appears to function as specific inhibitors of the cyclin/cdk complexes (Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. and Elledge, S. J. (1993) Cell 75: 805-816; Polyak, K., Lee,M.-H., Erdjument-Bromage, H., Koff, A., Roberts, J. M., Tempst, P., and Massague, J. (1994) Cell 78: 59-66; Toyoshima, H. and Hunter, T. (1994) Cell 78: 67-74; Matsuoka, S., Edwards, M. C., Bai, C., Parker, S., Zhang, P., Baldini, A., Harper, J. W., and Elledge, S. J. (1995) Genes and Dev. 9: 650-662). The second family of the cdk inhibitors is called INK4 family proteins. The four members of this family, called p15, p16, p18, and p19, bind directly to cdk4 and cdk6, and are therefore specific inhibitors of the cyclin D-dependent kinases (Hannon, G. J. and Beach, D. (1994) Nature 371: 257-261; Serrano, M., Hannon, G. J., and Beach, D. (1993) Nature 366: 704-707, Hirai, H., Roussel, M. F., Kato, J., Ashmun, R. A., and Sherr, C. J. (1995) Mol. Cell. Biol. 15: 2672-2681).
Although the precise roles of p27kip1 are far from clear, its level decreases when cells are stimulated to enter the cell cycle, and increases when cells are arrested by either the change in TGF-xcex2 concentration or contact inhibition (Polyak, K., Kato, J., Solomon, M. J., Sherr, C. J., Massague, J., Roberts, J. M., and Koff, A. (1994) Genes and Dev. 8: 9-22). p27kip1 was cloned as a binding protein with cyclin E-cdk2 (Polyak, K., Lee, M.-H., Erdjument-Bromage, H., Koff, A., Roberts, J. M., Tempst, P., and Massague, J. (1994) Cell 78: 59-66) or cyclin D-cdk4 (Toyoshima, H. and Hunter, T. (1994) Cell 78: 67-74). p27kip1 inhibits the activity of most cyclin-cdk complexes and can inhibit the phosphorylation of cyclin-cdk complexes by CAK (cdk-activation kinases) (Kato. J., Matsuoka, M., Polyak, K., Massague, J., and Sherr, C. J. (1994) Cell 79: 487-496). Therefore, p27kip1 functions as a negative regulator of the G1/S progression.
Tumor-specific mutations of the p27kip1 gene are rare, whereas several cell cycle regulators, such as p16, p53, and pRB, are frequently mutated in some cancers and have been shown to be tumor suppressor genes (Ponce-Castaneda, M. V., Lee, M.-H., Latres, E., Polyak, K., Lacombe, L., Montgomery, K., Mathew, S., Krauter, K., Sheinfeld, J., Massague, J., and Cordon-Cardo, C. (1995) Cancer Res. 55: 1211-1214). However, the p27kip1-deficient mice were observed to have increased body size, multiple organ hyperplasia, retinal dysplasia, and formation of pituitary tumors (Fero, M. L., Rivkin, M., Tasch, M., Porter, P., Carow, C. E., Firpo, E., Polyak, K., Tsai, L.-H., Broudy, V., Perlmutter, R. M., Kaushansky, K., and Roberts, J. M. (1996) Cell 85: 733-744; Kiyokawa, H., Kineman, R. D., Manova-Todorova, K. O., Soares, V. C., Hoffman, E. S., Ono, M., Khanam, D., Hayday, A. C., Frohman, L. A., and Koff, A. (1996) Cell 85: 721-732; Nakayama, K., Ishida, N., Shirane, M., Inomata, A., Inoue, T., Shishido, N., Horii, I., Loh, D. Y., and Nakayama, K. (1996). Cell 85: 707-720). These data are in part similar to the case of the RB heterozygous knockout mice (Hu, N., Gutsmann, A., Herbert, D. C., Bradley, A., Lee, W.-H., and Lee, E. Y.-H. P. (1994) Oncogene 9:1021-1027). In addition, it has been shown that lower expression of the p27kip1 protein correlated with poorer survival in breast cancer and colorectal cancer (Porter, P. L., Malone, K. E., Heagerty, P. J., Alexander, G. M., Gatti, L. A., Firpo, E. J., Daling, J. R., and Roberts, J. M. (1997) Nature Medicine 3: 222-225; Catzavelos, C., Bhattacharya, N., Ung, Y. C., Wilson, J. A., Roncari, L., Sandhu, C., Shaw, P., Yeger, H., Morava-Protzner, I., Kapsuta, L., Franssen, E., Pritchard, K. I., and Slingerland, J. M. (1997) Nature Medicine3: 227-230; Loda, M., Cukor, B., Tam, S. W., Lavin, P., Fiorentino, M., Draetta,.G. F., Jessup, J. M., and Pagano, M. (1997) Nature Medicine 3: 231-234). These results clearly indicate that p27kip1 plays an important role in inhibiting tumor formation and tumor progression. There have also been reports on the importance of the p27kip1 gene in enhancing the susceptibility of tumor cells to anticancer drugs and in influencing the prognosis factors of cancers (Croix, B. S., Florenes, V. A., Rak, J. W., Flanagan, M., Bhattacharya, N., Slingerland, J. M., and Kerbel, R. S. (1996) Nature Medicine 2: 1204-1210; Loda, M., Cukor, B., Tam, S. W., Lavin, P., Fiorentino, M., Draetta, G. F., Jessup, J. M., and Pagano, M. (1997) Nature Medicine 3: 231-234; Hengst, L. and Reed, S. I. (1996) Science 271: 1861-1864; Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., Sal, G. D., Chau, V., Yew, P. R., Draetta, G. F., and Rolfe, M. (1995) Science 269: 682-685). Consequently, it has been desired to develop drugs that regulate the transcription of the p27Kip1 gene in order to prevent or treat malignant tumors.
Recent reports showed that p27kip1 mRNA is induced by vitamin D3 in U937 cells (Liu, M., Lee, M.-H., Cohen, M., Bommakanti, M., and Freedman, L. P. (1996) Genes and Dev. 10: 142-153) and by neuronal differentiation (Poluha, W., Poluha, D. K., Chang, B., Crosbie, N. E., Schonhoff, C. M., Kilpatrick, D. L., and Ross, A. H. (1996) Mol. Cell., Biol. 16: 1335-1341). These facts suggest that the transcriptional regulation of the p27kip1 gene might also be important in cellular differentiation.
An object of the present invention is to provide the promoter of the human p27kip1 gene and a method of screening a compound capable of regulating the activity of the promoter.
The present inventors earnestly studied to achieve the above object. As a result, the inventors succeeded in isolating an upstream region of the human p27kip1 gene by preparing a partial fragment of the p27kip1 cDNA and screening a human leukocyte genomic library using this fragment as a probe. Furthermore, the inventors succeeded in identifying the basal promoter activity region within the upstream region by preparing deletion mutants of the upstream region and detecting their promoter activities. In addition, the inventors found that it is possible to screen compounds capable of regulating the promoter activity by using the isolated promoter region.
Namely, the present invention relates to a promoter region of the human p27kip1 gene and a method of screening a compound using the promoter region. More specifically, the invention relates to:
(1) a DNA comprising at least part of the nucleotide sequence of SEQ ID NO:1;
(2) a DNA comprising at least part of the nucleotide sequence of SEQ ID NO:1 and having the promoter activity;
(3) a vector comprising the DNA of (2) above;
(4) a cell carrying the vector of (3) above;
(5) a method of screening a protein capable of regulating the promoter activity of the DNA of (2) above, which comprises the steps selected from:
(a) steps of bringing a test sample into contact with the DNA of (2) above and selecting a protein that binds to the DNA of (2) above;
(b) steps of introducing a test DNA into cells carrying a reporter gene fused downstream of the DNA of (2) above and selecting an expression product that regulates the reporter gene expression; and
(c) steps of bringing a test sample into contact with cells carrying a reporter gene fused downstream of the DNA of (2) above and selecting a protein that regulates the reporter gene expression;
(6) a method of screening a DNA encoding a protein capable of regulating the promoter activity of the DNA of (2) above, which comprises the steps selected from:
(a) steps of bringing an expression product of a test DNA into contact with the DNA of (2) above and selecting a DNA encoding a protein that binds to the DNA of (2) above;
(b) steps of introducing a test DNA into cells carrying a reporter gene fused downstream of the DNA of (2) above and selecting a DNA encoding an expression product that regulates the reporter gene expression; and
(c) steps of bringing an expression product of a test DNA into contact with cells carrying a reporter gene fused downstream of the DNA of (2) above and selecting a DNA encoding an expression product that regulates the reporter gene expression;
(7) a method of screening a compound capable of regulating the promoter activity of the DNA of (2) above, which comprises the steps selected from:
(a) steps of bringing a test sample into contact with the DNA of (2) above in the presence of a test compound and selecting a compound that promotes or inhibits the binding between the DNA of (2) above and a protein in the test sample; and
(c) steps of bringing a test compound into contact with cells carrying a reporter gene fused downstream of the DNA of (2) above and selecting a compound that regulates the reporter-gene expression;
(8) a protein capable of regulating the promoter activity of the DNA of (2) above;
(9) the protein of (8) above, which can be isolated by the method according to (5) above;
(10) a DNA encoding a protein that regulates the promoter activity of the DNA of (2) above;
(11) the DNA of (10) above, which can be isolated by the method according to (6) above;
(12) a compound capable of regulating the promoter activity of the DNA of (2) above; and
(13) the compound of (12) above, which can be isolated by the method according to (7) above.